Erythropoietin is a naturally-occurring glycoprotein hormone with a molecular weight that was first reported to be approximately 39,000 daltons (T. Miyaki et al., J. Biol. Chem. 252:5558-5564 (1977)). The mature hormone is 166 amino acids long and the "prepro" form of the hormone, with its leader peptide, is 193 amino acids long (F. Lin, U.S. Pat. No. 4,703,008). The mature hormone has a molecular weight, calculated from its amino acid sequence, of 18,399 daltons (K. Jacobs et al., Nature 313:806-810 (1985); J. K. Browne et al., Cold Spring Harbor Symp. Quant. Biol. 51:693-702 (1986)).
Structural characterization of human urinary erythropoietin has identified a des-Arg166 form that results from specific removal of the Arg residue at the carboxy-terminus of the mature protein (M. A. Recny et al., J. Biol. Chem. 262:17156-17163 (1987)). Recny et al., supra, propose that the physiologically active form of erythropoietin circulating in human plasma is the des-Arg166 form.
Human erythropoietin contains three N-linked carbohydrate chains (H. Sasaki et al., J. Biol. Chem. 262:12059-12076 (1987); E. Tsuda et al., Biochemistry 27:5646-5654 (1988); and M. Takeuchiet al., J. Biol. Chem. 263:3657-3663 (1988)). The carbohydrate content of erythropoietin is similar in both naturally-occurring urinary erythropoietin and in hormone produced by expression, in mammalian cells in culture, of a cloned DNA which has been transfected into the cells and which encodes the prepro form of the hormone. The N-linked glycosylation sites are located at amino acid residues 24, 38, and 83. Both urinary and recombinant erythropoietin also contain a single O-linked glycosylation site at amino acid residue 126 (H. Sasaki et al., supra; E. Tsuda et al., supra; M. Takeuchi et al., supra; and M. Goto et al., Biotechnology 6:67-71 (1988)). The carbohydrate content of erythropoietin is a complex fucosylated tetra antennary type chain with and without N-acetyllactoseamine repeating units (M. Takeuchi et al., supra) and contributes approximately 40% of the mass of erythropoietin.
Human erythropoietin is primarily produced as a glycoprotein hormone by the adult kidney (H. P. Koeffler and E. Goldwasser, Ann. Intern. Med. 97:44-47 (1981)). The cells that produce erythropoietin in the kidney are rare and are located in the inner cortex of the renal parenchyma in the intersticium between renal tubules (S. T. Koury et al., Blood 71:524-528 (1988), and C. Lacombe et al., J. Clin. Invest. 81:620-623 (1988)). Consequently, destruction of kidney tissue, as occurs in renal failure, results in decreased production of erythropoietin and a concomitant reduction in erythrocyte count and anemia.
While fetal liver cells in vitro can produce erythropoietin (A. Kurtz et al., Endocrinology 118:567-572 (1986)), no compensating erythropoietin production occurs in most end-stage renal failure patients, and serum erythropoietin levels are normally restored only after successful renal transplantation (W. F. Denny et al., J. Lab. Clin. Med. 67:386 (1966)).
Late stage erythropoiesis, in most cases, is accomplished by a single glycosylated hormone, erythropoietin, produced in a single tissue. A rare alternate route of erythropoiesis has been documented in a human anephric patient with a high hematocrit. The isolated erythrotropic factor in this patient has been shown to be human insulin-like growth factor 1, or IGF-I (A. Brox et al., Exp. Hematol. 17:769-773 (1989), and L. F. Congote et al., J. Clin. Endocrin. Metab. 72:727-729). IGF-I is undoubtedly the human counterpart of the bovine erythrotropic factor described as having both in vivo and in vitro activity by L. F. Congote (Biochem. Biophys. Res. Comm. 115:477-483 (1983)). IGF-I receptors are known to exist on human erythrocytes, and these receptors could allow this rare alternate route of late stage erythropoiesis to occur via interaction of IGF-I and its specific receptor (T. Izami et al., J. Clin. Endocrinol. Metab. 62:1206-1212 (1986), and C. D. Costigan et al., Clin. Invest, Med. 11:4751 (1988)). However, the nucleotide sequence of the erythropoietin receptor gene is known and shows no sequence homology to that of the human IGF-I receptor (A. D. D'Andrea et al., Cell 57:277-285 (1989)), indicating that the alternate route of erythropoiesis via IGF-I is unrelated to the erythropoietin-mediated pathway of late stage erythropoiesis.
While no other alternate routes of late stage erythropoiesis are known, several factors have been described that can potentiate the action of erythropoietin. Late stage erythropoiesis is dependent on erythropoietin but is influenced by testosterone, estrogens, and erythroid-potentiating factor, while the early stage of erythropoiesis is dependent on burst-promoting activity in addition to erythropoietin (N. N. Iscove in Hematopoietic Cell Differentiation, eds. D. W. Golde, M. J. Cline and C. F. Fox Academic Press, New York! pp. 37-52). Factors such as IL-3, granulocyte macrophage colony-stimulating factor and interleukin-9 are known to have burst-forming activity. However, it is unclear whether these activities have any physiological role in erythropoiesis (J. Suda et al., Blood 67:1002-1006 (1986); C. A. Sieff et al., Science 230:1171-1173 (1985); and R. E. Donahoe et al., Blood 75:2271-2275 (1989). Recently, a factor termed "erythroid differentiation factor" has been shown to potentiate the activity of erythropoietin in vivo and in vitro (H. E. Broxmeyer et al., Proc. Natl. Acad. Sci. 85:9052-9056 (1988); J. Yu et al., Nature 330:765-767 (1987)). This factor has been shown to be identical to activin A (follicle-stimulating hormone-releasing protein) and to be inhibited by follistatin, a specific inhibitor of activin A; however, the physiological role of activin A remains to be determined (M. Shiozaki et al., Proc. Natl. Acad. Sci. 89:1553-1556 (1992)). Thus, after nearly twenty years of investigation, there is no clear indication that erythropoiesis is controlled by any hormone other than erythropoietin.
In the absence of any alternative hormones which affect erythropoiesis, several attempts to both probe erythropoietin structure and significantly improve the characteristics of erythropoietin by site-directed mutagenesis have appeared in the literature. The molecular cloning of the human gene encoding erythropoietin reveals a DNA sequence coding for a preprohormone of 193 amino acids and a mature hormone of 166 amino acids. The availability of cloned DNA encoding the hormone and its precursor (i.e., the prepro form) provides the opportunity for mutagenesis by standard methods in molecular biology. See U.S. Pat. No. 4,703,008, supra.
The first mutant erythropoietins (i.e., erythropoietin analogs), prepared by making amino acid substitutions and deletions, have demonstrated reduced or unimproved activity. As described in U.S. Pat. No. 4,703,008, replacement of the tyrosine residues at positions 15, 49 and 145 with phenylalanine residues, replacement of the cysteine residue at position 7 with an histidine, substitution of the proline at position 2 with an asparagine, deletion of residues 2-6, deletion of residues 163-166, and deletion of residues 27-55 does not result in an apparent increase in biological activity. The Cys.sup.7 -to-His.sup.7 mutation eliminates biological activity. A series of mutant erythropoietins with a single amino acid substitution at asparagine residues 24, 38 or 83 show severely reduced activity (substitution at position 24) or exhibit rapid intracellular degradation and apparent lack of secretion (substitution at residue 38 or 183). Elimination of the 0-linked glycosylation site at Serinel26 results in rapid degradation or lack of secretion of the erythropoietin analog (S. Dube et al., J. Biol. Chem. 33:17516-17521 (1988)). These authors conclude that glycosylation sites at residues 38, 83 and 126 are required for proper secretion and that glycosylation sites located at residues 24 and 38 may be involved in the biological activity of mature erythropoietin.
The suggestion that glycosylation of erythropoietin is required for in vitro biological activity is contrary to reports showing that deglycosylatjed erythropoietin is fully active in vitro bioassays (M. S. Dordal et al., Endocrinology 116: 2293-2299 (1985); J. K. Browne et al., Cold Spring Harbor Symp. Quan. Biol. 51:693-702 (1986); U.S. Pat. No. 4,703,008; E. Tsuda et al., Eur. J. Biochem. 188:405-411 (1990); and K. Yamaguchi, et al., J. Biol. Chem. 266:20434-20439 (1991)). A set of analogs of erythropoietins, similar to those studied by Dube et al., supra, has been constructed using oligonucleotide-directed mutagenesis to probe the role of glycosylation sites in the biosynthesis and biological activity of erythropoietin (K. Yamaguchi et al., supra). These investigators conclude that glycosylation is important for the correct biosynthesis and secretion of erythropoietin but has no affect on the in vitro activity of the molecule. However, all of the mutant erythropoietins studied by Yamaguchi et al., which involve changes at the glycosylation sites, lack in vivo biological activity.
Glycosylation of erythropoietin is widely accepted to play a critical role in the in vivo activity of the hormone (P. H. Lowy et al., Nature 185:102-105 (1960); E. Goldwasser and C. K. H. Kung, Ann. N.Y. Acad. Science 149:49-53 (1968); W. A. Lukowsky and R. H. Painter, Can. J. Biochem. 50:909-917 (1972); D. W. Briggs et al., Amer. J. Phys. 201:1385-1388 (1974); J. C. Schooley, Exp. Hematol. 13:994-998; N. Imai et al., Eur. J. Biochem. 194:457-462 (1990); M. S. Dordal et al., Endocrinology 1 16:2293-2299 (1985); E. Tsuda et al., Eur. J. Biochem. 188:405-411 (1990); U.S. Pat. No. 4,703,008; J. K. Brown et al., Cold Spring Harbor Symposia on Quant. Biol. 51:693-702 (1986); and K. Yamaguchi et al., J. Biol. Chem. 266:20434-20439 (1991)).
The lack of in vivo biological activity of deglycosylated analogs of erythropoietin is attributed to a rapid clearance of the deglycosylated hormone from the circulation of treated animals. This view is supported by direct comparison of the plasma half-life of glycosylated and deglycosylated erythropoietin (J. C. Spivak and B. B. Hoyans, Blood 73:90-99 (1989), and M. N. Fukuda, et al., Blood 73:84-89 (1989).
Oligonucleotide-directed mutagenesis of erythropoietin glycosylation sites has effectively probed the function of glycosylation but has failed, as yet, to provide insight into an effective strategy for significantly improving the characteristics of the hormone for therapeutic applications.
A series of single amino acid substitution or deletion mutants have been constructed, involving amino acid residues 15, 24, 49, 76, 78, 83, 143, 145, 160, 161, 162, 163, 164, 165 and 166. In these mutants are altered the carboxy terminus, the glycosylation sites, and the tyrosine residues of erythropoietin. The mutants have been administered to animals while monitoring hemoglobin, hematocrit and reticulocyte levels (European Published Patent Application No. 0 409 113). While many of these mutants retain in vivo biological activity, none show a significant increase in their ability to raise hemoglobin, hematocrit or reticulocyte (the immediate precursor of an erythrocyte) levels when compared to native erythropoietin.
Another set of mutants has been constructed to probe the function of residues 99-119 (domain 1) and residues 111-129 (domain 2) (Y. Chern et al., Eur. J. Biochem. 202:225-230 (1991)). The domain 1 mutants are rapidly degraded and inactive in an in vitro bioassay while the domain 2 mutants, at best, retain in vitro activity. These mutants also show no enhanced in vivo biological activity as compared to wild-type, human erythropoietin. These authors conclude that residues 99-119 play a critical role in the structure of erythropoietin.
The human erythropoietin molecule contains two disulfide bridges, one linking the cysteine residues at positions 7 and 161, and a second connecting cysteines at positions 29 and 33 (P.-H. Lai et al., J. Biol. Chem. 261:3116-3121 (1986)). Oligonucleotide-directed mutagenesis has been used to probe the function of the disulfide bridge linking cysteines 29 and 33 in human erythropoietin. The cysteine at position 33 has been converted to a proline residue, which, mimics the structure of murine erythropoietin at this residue. The resulting mutant has greatly reduced in vitro activity. The loss of activity is so severe that the authors conclude that the disulfide bridge between residues 29 and 33 is essential for erythropoietin function (F.-K. Lin, Molecular and Cellular Aspects of Erythropoietin and Erythropoiesis, pp. 23-36, ed. I. N. Rich, Springer-Verlag, Berlin (1987)).
Site-specific oligonucleotide-directed mutagenesis of the methionine residue at position 54 of human erythropoietin results in a molecule which retains the in vivo biological activity of the parent (wild-type) molecule with the added advantage of providing an erythropoietin preparation which is less susceptible to oxidation (Shoemaker, U.S. Pat. No. 4,835,260).
A large number of mutants of the human erythropoietin gene have been described in several scientific publications and patent applications. These mutants have spanned the entire length of the molecule, have produced partially- or completely-deglycosylated molecules, have altered the structures of the disulfide bridges in the molecule, and have attempted to improve the therapeutic activity of the molecule. Of all such attempts to alter erythropoietin, none have succeeded in producing a molecule with enhanced in vivo biological activity or other improved properties for therapeutic applications.
The failure to identify a naturally-occurring alternate route of late stage erythropoiesis and the heretofore unsuccessful attempts to produce an erythropoietin analog with enhanced in vivo activity have provided little insight into how an improved erythrotropic molecule could be made.